Band 3 is the major membrane spanning protein of the human erythrocyte, present at one million copies/cell, where it functions to mediate the exchange of chloride and bicarbonate across the lipid bilayer. The NH2-terminal 43,000 daltons of the protein forms a hydrophilic domain at the cytoplasmic surface of the red cell membrane which can be isolated by mild proteolytic digestion. This portion of the protein serves as an important attachment point for cytoskeleton to the erythrocyte membrane as well as a binding site for hemoglobin and several of the glycolytic enzymes within the red cell. The principal goals of the research proposed in this application are: 1) to determine the structure of the cytoplasmic domain of band 3; 2) to characterize the nature of the interaction of hemoglobin and the glycolytic enzymes with band 3; 3) to clone and sequence the cDNA for band 3; and 4) to use oligonucleotide probes, and later the cDNA, to prove a human genomic library for the gene for band 3 and hemologous proteins which may interact with the cytoskeleton in other tissues. Recently, we have directly shown that the acidic NH2-terminal sequence of band 3 is the binding site for hemoglobin. A synthetic peptide corresponding to the first 11 residues of band 3, AcM-E-E-L-Q-D-D-Y-E-D-E, as well as the intact cytoplasmic domain, were found to bind preferentially to deoxyhemoglobin. X-ray studies of the deoxyhemoglobin-peptide complex showed that the peptide binding site includes the binding site for 2,3-diphosphoglycerate (DPG) and, in addition, extends deeply 18 angstroms into the central cavity between the Beta subunits. Further experiments are proposed to determine the relative affinity of deoxyhemoglobin and several of the glycolytic enzymes for band 3 under physiological conditions, to determine the effect of pH, ionic strength, and competing metabolites (e.g. DPG) on the hemoglobin-band 3 interaction, to image the stereochemistry of the peptide-Beta chain contacts at high resolution, and finally to crystallize and determine the structure of the cytoplasmic domain of band 3. Our studies of the molecular biology of band 3 are aimed at pursuing the hypothesis, suggested by immunological studies, that membrane proteins in other tissues may interact with the cytoskeleton through segments of the polypeptide homologous with the cytoplasmic portion of band 3. These studies also will provide the basis for investigation of the regulated expression of band 3 during erythroid development.